The body of this research program has focused on the critical relationship between lipids and cellular proteostasis of the presynaptic protein alpha-synuclein (alpha-syn) and how it relates to Parkinson disease (PD). A fundamental question that is addressed is the role of membranes in promoting misfolding and aggregation of alpha-syn. We have carried out detailed investigations of membrane interactions and amyloid formation of alpha-syn that have provided residue-specific information and molecular insights into the mechanism of aggregation. Due to the complexity of the amyloid problem, the tools with which we attack have included molecular biology, steady-state and time-resolved fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, electron microscopy, neutron reflectometry (NR), and mass spectrometry. We are developing a chemical understanding in how specific phospholipids modulate protein structure, membrane binding, and aggregation propensity through many different studies summarized below. Further, we are moving towards translating sophisticated spectroscopic measurements into a cellular context as well as designing biochemical analyses of whole tissue lysates. We are continuing our efforts in pushing the utility of NR, a scattering method, to investigate membrane interactions of alpha-syn. NR is unique among available structural techniques in that it allows the characterization of membrane-associated proteins bound to actual lipid bilayers in an aqueous environment, as opposed to proteins in solid state or detergent solubilized forms currently prevalent in the field. The reflection of neutrons from bilayer membranes simultaneously probes the protein and the bilayer and readily distinguishes the layers of lipid acyl chains and phospholipid headgroups as well as the membrane bound proteins. Both the extent of polypeptide insertion into the bilayer and extension into the aqueous surrounding can be measured. However, NR alone cannot directly provide spatial resolution of specific residues within the bilayer so that information must be inferred from other experimental methods. To address this limitation, we produced segmentally deuterated alpha-syn via native chemical ligation (NCL). The ligation of the uniformly deuterated portion (1-86 or 87-140) to the respective protiated segment (87-140 or 1-86) provided scattering contrast within the full length -syn. By using segmentally deuterated alpha-syn, we have successfully identified region-specific protein-membrane interactions by NR. The coupling of NCL and NR has never been demonstrated before. This combined method allowed the identification of specific region of protein-membrane interaction in a fluid, tethered bilayer. This strategy applies generally to other studies of membrane protein folding. Similarly, the NCL protocols developed here are useful for other techniques that are sensitive to isotope substitutions such as NMR, Raman, and Fourier transform infrared spectroscopy. Recent work from my lab indicates that alpha-syn strongly influences the structure and properties of phospholipid bilayers including membrane thinning and formation of lipid tubular structures. Other examples of alpha-syn-lipid structures such as bilayer discs and micellar tubes also have been reported in the literature. We are interested in the interplay between protein structure and membrane deformation and how this process is regulated by phospholipid composition. In mice with all three alpha-, beta-, and gamma-syn genes knocked out, enhanced levels of several BAR domain proteins, membrane curvature sensing/generating proteins, were enhanced, implying up-regulation to compensate the loss of function from synuclein deficiency. We are motivated by these observations and aim to elucidate the mechanism of phosphatidylcholine membrane bending and tubulation by alpha-syn as it is especially pertinent in the cellular environment where such action can have devastating consequences. In relating to the complex cellular lipid compositions, we are focusing towards understanding the effects of bilayer fluidity and phase state by changes in acyl chain length as well as chain saturation. Results from these studies will establish a firm basis in supporting our cellular work. An important focus for the future is to study conformational dynamics and aggregation of alpha-syn in a cellular context. While broadly used in vitro, standard protein secondary structural determination techniques such as CD, NMR, and FTIR spectroscopy are ill-suited to make direct observations of protein conformational changes and amyloid formation in cellular systems. To address the need, we are developing Raman microscopy as an alternate approach for characterizing amyloid formation of alpha-syn in cellular environments. This direct spectroscopic method reports on intrinsic molecular vibrations such as protein amide bonds, which arise from coupled vibrational modes of the polypeptide backbone. The position and widths of the amide band peaks depend on the peptide-bond angles and hydrogen-bonding patterns, and therefore, inform on protein secondary structure as well as local environment. Conformations of alpha-helix, beta-sheet, or random coil exhibit characteristic peak maxima, making quantification of structural compositions possible. By coupling the chemical specificity of Raman spectroscopy and the spatial resolution of a microscope, we will gain molecular level information on the cellular fate of alpha-syn by directly evaluating the development of beta-sheet structure, while simultaneously pinpointing its location and thus, identify initiation sites of amyloid formation.